Amplification and/or overexpression of erbB2 and erbB3 occurs in a significant number of human breast and ovarian carcinomas. Heregulin (HRG) has been demonstrated to bind to cells coexpressing ErbB2 and ErbB3. These two receptors and the epidermal growth factor receptor (EGFR) possess substantial homology, and HRG contains a 68 amino acid EGF-like domain. Despite these similarities, binding of EGF to cells coexpressing ErbB2 and ErbB3 has not been reported. The interleukin (IL)-3-dependent 32D line does not endogenously express any ErbB receptor family member. Here we report that EGF is able to specifically bind to and induce mitogenesis, long-term proliferation and tyrosine phosphorylation events in 32D transfectants coexpressing ErbB2 and ErbB3 but not in transfectants expressing either receptor individually. Unlike EGF, transforming growth factor-alpha (TGF-alpha) is unable to mediate efficient mitogenesis in the 32D cotransfectant. EGF is also shown to induce receptor tyrosine phosphorylation in MDA-MB134 human breast carcinoma cells that express ErbB2 and ErbB3 but not EGFR. These results provide evidence that EGF is a ligand for cells that co-express ErbB2 and ErbB3 and may contribute to the progression of carcinomas expressing these two receptors.